Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

Zetina, Carlos R.; Goldberg, Michel

doi:10.1016/0022-2836(82)90516-2

Cited by 56 publications

(32 citation statements)

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“…Each monomer is formed by two autonomously folding structural domains [8,9]. In the models presented here we cannot discern either the 8 protomers or the individual domains.…”

Section: Model Calculationsmentioning

confidence: 99%

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Wilhelm

Pilz

Lane

et al. 1982

European Journal of Biochemistry

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The a and 0 2 subunits of tryptophan synthase were investigated by small-angle X-ray scattering. The molecular parameters are: radius of gyration, CI: 1.95 nm, pz : 3.01 nm; maximum particle diameter, CI: 5.8 nm, p 2 : 10.5 nm; and hydrated volume, CI : 60 nm3, p2 160 nm'. The shape of the CI subunit can best be described by a circular cylinder, slightly tapered at one end. An elongated elliptical cylinder with its cross section larger in the middle than at the ends was found to be a model equivalent in scattering to the p2 subunit.The CI& enzyme complex was found to have a radius of gyration of 4.01 nm, a maximum length of 13.5 nm, and a hydrated volume of 270 nm3. No satisfactory fit of the scattering data was obtainable by mere apposition of the models of the CI and p 2 subunits. Two cylinders overlapping laterally fit the experimental data considerably better, suggesting changes in the conformation of the subunits on forming the CI& complex.Tryptophan synthase from Escherichia coli is an CI& bienzyme complex, which catalyses the following reactions :Reaction (1) is catalyzed by the CI subunit and 100-fold more efficiently by the (x& complex. Reaction (2) is catalyzed by the fl2 subunit and 50-fold more efficiently by the complex. This mutual activation is thought to arise from heterologous subunit-subunit interactions during assembly of the complex. Moreover, the active sites appear to be juxtaposed at the intersubunit interface [l].The information on the structure and function of the complex and the component subunits has been reviewed recently [I]. Meanwhile the nucleotide sequences of the corresponding genes have been determined for E. roli [2], other enteric bacteria [3], and for Saccharomyces cerevisiae [4], showing that the amino acid sequences of the CI and / 3 polypeptide chains are highly conserved. Further, limited proteolysis has revealed that both the (x subunit ( M , 28727) and the p protomer (M, 42988) are composed of two autonomously folding structural domains that probably form the corresponding active sites at the interdomain interfaces [5 -91. Except for a preliminary crystallographic study of the a subunit [lo] and an estimate of the distance between the two active sites [Ill, little direct evidence on the tertiary and quaternary structure of tryptophan synthase and its subunits is available to date.In this paper we present the results of small-angle X-ray scattering studies on the CI and p2 subunits and the a& complex of tryptophan synthase. The data were fitted to various models equivalent in scattering and support a scheme for the assembly of the complex from its subunits. MATERIALS AND METHODS EnzymesTryptophan synthase (x-& complex was purified from the overproducing strain Escherichia coli trpR trpAEDl02/ F'trpAEDl02 [12]. The excess CI subunit produced by this strain was purified by affinity chromatography [13]. The p 2 subunit was prepared from the pure CI& complex by heat denaturation as described [12,14]. Protein aggregates were removed by gel chromatography on Sephacryl S200 (...

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“…Each monomer is formed by two autonomously folding structural domains [8,9]. In the models presented here we cannot discern either the 8 protomers or the individual domains.…”

Section: Model Calculationsmentioning

confidence: 99%

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Wilhelm

Pilz

Lane

et al. 1982

European Journal of Biochemistry

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“…These results suggest that the dimer DЈ is greatly expanded, having disordered segments at the amino terminus and between domains 1 and 2. For most oligomeric proteins, forming dimers occurs only after the formation of nearly native monomers (18,19). This does not seem to be the case with apo-eSHMT.…”

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confidence: 99%

Structural Studies on Folding Intermediates of Serine Hydroxymethyltransferase Using Single Tryptophan Mutants

Cai

Schirch

1996

Journal of Biological Chemistry

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Previous studies showed that during the in vitro folding of Escherichia coli serine hydroxymethyltransferase at 4°C, both monomer and dimer intermediates accumulated and were stable for periods of minutes to hours (Cai, K., Schirch, D., and Schirch, V. (1995) J. Biol. Chem. 270, 19294 -19299). To obtain structural information on these intermediates, two of the three Trp residues in the protein were changed to Phe to generate a set of three single Trp mutant enzymes. These mutant enzymes were purified and characterized and shown to retain essentially all of the properties of the wild-type enzyme. The fluorescence and circular dichroism measurements of each mutant enzyme were studied under unfolding-refolding equilibrium conditions and during refolding. In addition, the sensitivity of the protein to digestion by subtilisin during refolding was investigated. The results of these studies show that the unfolded enzyme has two domains that rapidly fold to form a monomer in which the first 55 amino acids and a segment between residues 225 and 276 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate determining conformational change in which the unstructured segments assume their native state.Escherichia coli serine hydroxymethyltransferase (eSHMT) 1 is a 94-kDa homodimer that catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one carbon carrier. Each subunit of the holoenzyme contains PLP attached to Lys 229 as an aldimine. Removal of the PLP generates the apoenzyme, which remains dimeric. The 417-residue subunit contains 3 Trp residues at positions 16, 183, and 385 (1).In a previous study we demonstrated that eSHMT could be reversibly refolded from 8 M urea after a 10-fold dilution at concentrations as high as 1 mg/ml of protein (2). At 30°C refolding was essentially complete in 6 min. This study also provided evidence for several intermediates and a mechanism as shown in Equation 1. The most important information was obtained with refolding done at 4°C, where distinct kinetic intermediates could be shown to exist for periods of several hours and complete refolding took as long as 20 h. U is the unfolded enzyme and rapidly folds in a few seconds to form a monomer M. This monomer forms dimers (labeled as DЈ) during 20 min at 4°C. The dimer DЈ does not bind PLP but undergoes a slow rate-determining conformational change to a dimer (apoD) that can bind PLP to form native holoD. At 30°C the process of converting DЈ to apoD takes a few minutes, but at 4°C it takes many hours.The purpose of this study was to obtain structural information about M, DЈ, and apoD. Because these intermediates exist for minutes to hours at 4°C, a variety of physical measurements can be made to aid in elucidation of their structure. Important information can be obtained on the structure of intermediates by monitoring the fluorescence properties of Trp residues. To simplify the interpretation of results with eSHMT, we made single Trp mutants ...

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“…Reduced holo-fl2 was obtained by sodium borohydride reduction of holo-~'2 [15]. fl2-AEDANS was prepared as described by Zetina and Goldberg [8].…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, 0.1 M GdnHC1 does not seem to affect significantly the kinetics of refolding of #2 [8]. We therefore chose to study the refolding of #2 by reducing the GdnHC1 concentration from 4 M to 0.1 M (or below) in a stopped-flow.…”

Section: Determination Of Mixing Conditionsmentioning

confidence: 99%

“…In this field, one of the most extensively characterized systems is the b'2-subunit of Escherichia coli tryptophan synthase. Previous reports from this laboratory described the existence of 6 intermediates on the folding pathway [7][8][9][10][11]. According to this result, b-chains renature at 12°C according to the following minimal scheme: It can be seen that the rate constants of/~IR and /fI1 appearances are too rapid to be conveniently measured by conventional manual mixing methods, even at 12°C where the folding process is already slowed down as compared to 37°C (the physiological temperature).…”

Section: Introductionmentioning

confidence: 95%

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Renaturation of guanidine‐unfolded tryptophan synthase by multi‐mixing stopped‐flow dilution in D₂O

1988

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Guanidine hydrochloride (GdnHC1) at high concentrations, e.g. 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins. The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 5~100-fold in a 'renaturation buffer'. Fast kinetic studies, using a stopped-flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing. Secondly, the density differences existing between the concentrated GdnHCI solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped-flows. Here, it is shown that these difficulties can be overcome by using a multi-mixing stopped-flow to achieve 2 successive 7-fold dilutions and by using heavy water (D20) to adjust the density of the renaturation buffer. This enabled us to study the appearance of a short-lived intermediate during the folding of the fl2-subunit of Escherichia coil tryptophan synthase.

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Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

Cited by 56 publications

References 20 publications

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Structural Studies on Folding Intermediates of Serine Hydroxymethyltransferase Using Single Tryptophan Mutants

Renaturation of guanidine‐unfolded tryptophan synthase by multi‐mixing stopped‐flow dilution in D₂O

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Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

Cited by 56 publications

References 20 publications

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β2 Subunits

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β2 Subunits

Structural Studies on Folding Intermediates of Serine Hydroxymethyltransferase Using Single Tryptophan Mutants

Renaturation of guanidine‐unfolded tryptophan synthase by multi‐mixing stopped‐flow dilution in D2O

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Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Small‐Angle X‐Ray Scattering Studies of Tryptophan Synthase from Escherichia coli and Its α and β₂ Subunits

Renaturation of guanidine‐unfolded tryptophan synthase by multi‐mixing stopped‐flow dilution in D₂O